The goal of the work outlined in this grant is to understand the role of mitogen-activated protein kinase (MAPK) in smooth muscle contractility. During smooth muscle contraction, MAPK is activated, co-localizes with actin, and phosphorylates the thin filament protein caldesmon (CaD). Although the only smooth muscle substrate for MAPK identified thus far os CaD, the functional biochemical effects of the phosphorylation remain controversial. In conflicting studies using permeabilized smooth muscles, Nixon, et.al., found that MAPK had no effect on contractility, while Gerthoffer, et.al., found that MAPK induced a contraction. In the current proposal, the fundamental hypothesis tested is that: MAPK activation and the subsequent phosphorylation of its substrates in the contractile apparatus results in altered smooth muscle contractility. This hypothesis will be studied by performing the following specific aims: Aim 1. To determine the biochemical effects of ERK1 phosphorylation in an aortic actomyosin preparation. Specifically, alterations in actomyosins ATPase activity due to ERK1, and unique substrates for ERK1 in the actomyosin domain, will be identified. Aim 2. To determine the contractile effects of ERK1 in permeabilized arterial smooth muscles. Effects of purified ERK1 on the calcium sensitivity of contraction will be tested. Aim 3. To characterize the binding of wild type (wt) ERK1 or a mutant ERK1 (ERK1k67R) to CaD in order to test the sub-hypothesis that ERK1k67R can act as a dominant negative MAPK in smooth muscle. Aim 4. To test the in vivo function of MAPK in vascular smooth muscle by eliminating MAPK activity in a mouse transgenic model system. Transgenic mice overexpressing ERK1k67R specifically in smooth muscle will be developed and measurements will be made of biochemical and psychological changes that occur in the animals. The mechanisms responsible for determining the level of force attained in smooth muscle are not completely understood and may involve MAPK. The proposed studies will identify the role of this kinase in regulating contraction, the primary function of smooth muscle.